Accumulation and breakdown of RNA-deficient intracellular virus particles in interferon-treated NIH 3T3 cells chronically producing Moloney murine leukemia virus.

(1)

0022-538X/83/020489-07$02.00/0

Accumulation and

Breakdown of RNA-Deficient Intracellular

Virus

Particles in Interferon-Treated NIH 3T3 Cells

Chronically Producing

Moloney

Murine

Leukemia Virus

MORDECHAI ABOUD* AND YEHUDITH HASSAN

Departmentof Microbiology & Immunology, Faculty of Health Sciences, Ben Gurion University of the

Negev,BeerSheva,Israel

Received 27 August1982/Accepted 13 October 1982

Interferon treatment of NIH 3T3 cells chronically infected with Moloney

murine leukemia virus inhibited about95%of virus release. This inhibition was

accompanied byathree-totwofold accumulation of intracellularvirions. Howev-er,this accumulation could be demonstratedonly be exogenous reverse

transcrip-tasereactionassay orradioactivelabeling of the assembled viral proteins.Itcould

not be shown by the endogenous reverse transcriptase reaction assay, which

dependedonendogenousviralRNA, orby labeling the encapsidated viralRNA.

It was therefore evident that most ofthe intracellular virions accumulated in

interferon-treated cellswere RNAdeficient. Hybridization analysisrevealedthat

thesevirions weredeficient of genomic viral RNA, whereas size analysis by gel

electrophoresis suggested that the deficiency of 4S RNA normally packaged in

Moloneymurine leukemia viruswas evenstronger. Our data alsosuggestedthat

thisRNAdeficiencywasnotdueto adegradation ofthe encapsidated RNA, but morelikely to adefect in virus assembly. RNA-lacking intracellularvirionswere

unstable; theywerefoundto collapse before beingreleased.

TypeC retroviruses areusuallyassembledat

the inner side of the plasma membrane and

finally released from the cell surface by a

bud-dingprocess (13, 20, 30).Therefore, completed

virionscanbe detected in suchcasesonly

extra-cellularly. However, there are reportsofafew

exceptions. Forinstance, Friend leukemia virus

particleshave beenillustratedbyseveral

investi-gators to exist in cytoplasmic vacuoles of

di-methyl sulfoxide-activated erythroleukemic

cells (19, 23, 28, 35-37), in whichthey seem to

be stored until theyare expelled byopening of the vacuoles at the cell surface (28). Early

studies have shown vacuolartypeCparticlesof

Moloneymurine leukemia virus (MLV)inbone

marrowcells of leukemicrats(18)andparticles

of Moloney murine sarcoma virus in cells of

solid tumors of mice (17). Recently, we have

shown, by transmission electron microscopy,

the existenceinlarge numbers ofMLVparticles

incytoplasmic vacuoles ofthe NIH 3T3cellline

(2a, 6)and provided indicationsto suggest that

theyareformedintracellularly(1),apparently by budding intothespaceofthecytoplasmic

vacu-oles,wheretheyarekept untiltheirrelease from

the cells(M.Aboud etal., submittedfor

publica-tion).Onlyrarevirusparticleshave beenseen to

bud in the usual way through the outer

mem-brane of these cells. Furthermore, we have shown thatinterferon (IFN) treatment strongly

inhibitsvirus releasefromthesecells,leadingto

a remarkable accumulation of intracellular

viri-ons(2a,34).Thisfindingis instrikingcontrast to

the effect of IFN on Friend leukemia

virus-producing cells observed by Luftig et al. (28).

These authors have shown that, whereas control cells containedmanyvacuolarvirions,the

cyto-plasmicvacuolesof IFN-treated cellswere

emp-ty of virus particles, suggesting that in their

systemIFNinhibits the formationof the

vacuo-larvirions rather than their release.

However, in many repeated experiments we

havenoticed that IFN-inducedaccumulationof intracellular virions in our system was much

more apparent in thin-section electron

micro-scopic examinations or by measuring parame-ters representing assembled viral proteins than bymeasuring parameters representing

encapsi-dated viral RNA. Therefore, in this study we

carried out a systematic investigation of the

significanceof thisinconsistency and concluded

that mostoftheintracellular virions

accumulat-ed in IFN-treataccumulat-ed cells were RNA deficient.

Moreover, we presentdatasuggestingthatafter

alimitedstorageperiodtheRNA-lackingvirions

collapse within the cells, whereas the

RNA-489

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

490 ABOUD AND HASSAN

containing virions seem to be rather stable and

apparently are released from the cells at a re-duced rate imposed by IFN.

MATERIALS AND METHODS

Cells and viruses. NIH 3T3mousefibroblasts chron-ically infected with MLV were used throughout this study. The cells were grown in Dulbecco modified Eagle mediumcontaining10% newborn calfserum.In experiments with IFN the serum concentration was reduced to2.5%.

IFN. Mouse IFN was prepared as described else-where (5). The IFN preparation used in this study contained 7x 106U/mgofproteinandwasappliedat a concentration of 80 U/ml.

Isolation of extracellular and intracellular virions. Extracellular virions were pelleted from the culture medium through a 20% sucrose cushion, after the medium was first cleared of cell debris. Thepelleted

virions were furtherpurifiedbybanding in a 15 to60%'o

sucrose gradient. To isolate intracellularvirions, the cells were thoroughly washed four times with cold

phosphate-bufferedsalinefor maximal removal of

re-sidualextracellular virions. The cellswerethen exten-sively treated withtrypsin-EDTAtofurtherdestroy any residue of extracellular virions. Although our previous electronmicroscopic studies (6; Aboud et al., in press) revealed that only rare virus particles were

externally associated with the outer membrane of

these cells,compared with thenumerousintracellular virions in cytoplasmic vacuoles, the trypsin-EDTA treatment was aimed also at removing these few external cell-associated virions, so that the isolated virusparticleswould be entirely of intracellular origin. Thecells werehomogenized,andapostmitochondrial

cytoplasmic fraction was prepared aspreviously

de-scribed(34). Thecytoplasmicfractionwastreated for 15 min with 5 mM EDTAtodissociatepolyribosomes

and further treated with 20 pLg of RNase A (Sigma

ChemicalCo.)perml for 30minat37°CtodigestRNA molecules which wereoutside of virusparticles. The

cytoplasmic virions werepelletedthrougha20%

su-crosecushion and then banded in 15 to60%sucrose

gradients. Unlabeled virionswerelocated in the

gradi-ent by reverse transcriptase assay ofeach fraction collected from the bottom of the gradient. Labeled virionswerelocatedbytrichloroaceticacid

precipita-tionofaliquots taken from each fraction of the

gradi-ent.

Reverse transcriptase assay. The exogenous reac-tion assay, using polyriboadenylate-oligothymidylate

[poly(rA) -oligo(dT)] as a template-primer, was

de-scribedby Aboudetal.(5). Theendogenous reaction assaywasperformedinthe same mannerexcept that poly(rA) -oligo(dT) wasomitted. In some

confirma-toryexperiments,75 ,ug ofactinomycinDper mlwas addedtotheendogenous reaction -mixture.

Radioactivelabeling. Cellswerelabeledwith 75,uCi

of[3H]uridine(50Ci/mmol; Nuclear Research Center, Negev, Israel)or 35 ,uCiof[3H]leucine 39Ci/mmol;

Nuclear ResearchCenter) perml.

Viral RNA extraction.Unlabeledorlabeledvirions, purified in a sucrose gradient, were lysed by 0.5% sodium dodecyl sulfate; RNA was extracted by a mixture of chloroform-isoamyl alcohol (24:1), and,

after50pLgof EscherichiacolitRNAper mlwasadded

as acarrier, RNA was concentrated by ethanol precip-itation.

Analysis ofgenomic RNA sequences. Concentrated viral RNA of unlabeled virions was centrifuged through a 15 to30% sucrose gradient; 3H-labeled 28S, 18S, and 4S RNA were used as size markers. Frac-tions were collected from the viral RNA-containing gradients, 50 ,ugof E. coli tRNA per ml was added to eachfraction, and RNA was precipitated from these fractions by ethanol and assayed for viral genomic RNA sequences by hybridization to MLV-specific

[3H]cDNAasdetailedby Salzberg et al. (34). By using purified 70S MLV RNA, this hybridization reaction was proved to belinear within a range of 15 to 65% hybridization.

Size analysis of viral RNA by gel electrophoresis. Equal amounts of[3H]uridine-labeled viral RNA (in termsof counts perminute) were boiled for 3 min with

0.5% sodium dodecyl sulfate and then analyzed by

1.5% agarose-1.8% polyacrylamide gel

electrophore-sis. The gel was sectioned into 2-mm slices, which wereextractedovernightby 0.5 mlof30%H202 and counted inInstagel scintillation liquid (Packard Instru-mentCo., Inc.), with correctionfor quenching of each sample.

RESULTS

Effect of IFN on reverse transcriptase activity

of intracellular virions. Virus-producing cells

were incubated for 48 h in the presence or

absence ofIFN. Extracellularvirions, released during this incubation period, were harvested from the culture medium and banded in 15 to

60% sucrosegradients. Each fraction collected

from thegradientswasassayed forreverse

tran-scriptase in exogenous reactions, using poly

(rA) * oligo(dT)asanexogenous template-prim-er for virus quantitation. IFN inhibited virus releases by about95% (Fig.1A).

TodeterminetheeffectofIFN on the intracel-lular virions under these conditions, the cells

were disrupted and intracellular virions were

isolated in 15 to 60% sucrose gradients. An

exogenous reverse transcriptase reaction assay

of the gradient fractions (Fig. 1B) revealed a

three- to fourfold-higher activity in the viral

band of IFN-treated cells than in that of the

control. However, when the enzyme activity

wasassayed inendogenousreactions,no

signifi-cantdifference was observed(Fig. 1C). Similar results were obtained if actinomycin D was

added to the endogenous reaction mixture to

avoid DNA-dependent DNA synthesis, thus

eliminating a possible argument that cellular

DNApolymerasesweremeasured in this

experi-ment. Apossibleexplanation for this

discrepan-cyisthat theincreasedactivity ofthe

intracellu-lar virions ofIFN-treated cells detected in the

exogenousreactionrepresents ahigher

accumu-lation of virus particles, but most of them are

probably deficient of viral RNA, which is

re-quired for measuring the enzyme activity in

J. VIROL.

http://jvi.asm.org/

(3)

J(L)

-x

Q~o

a-Jx cr

Z5J

(I)

-JQ

3r x

J- 2.

a: L

4 31

i.20 f

1.15H 1.10

c-1.05Z

H

0 4 8 12 16 20 24

FRACTION NUMABER

FIG. 1. Reversetranscriptase activity of extracel-lular and intracelextracel-lular virions of IFN-treated and

un-treated cells. Virus-producingcells weretreated with

80 U of IFNpermlfor 48 h(S).Untreated cells served

asacontrol(0).Extracellular virionswereharvested from the culture medium(A),and intracellular virions

were harvested from the postmitochondrial cytoplas-mic fractions of the cells (B and C). Virions were banded in 15 to 60% sucrose gradients. Fractions, collected from the gradients, were assayed for viral

reversetranscriptase activityinexogenous(AandB) andendogenous (C)reactions.

endogenous reactions. Another possible

expla-nation is based on the observation of

Bandyo-padhyay et al. (8) that IFN may interfere with

the formation ofapropercomplex between p30

andreversetranscriptase, which is important for

viral RNA-directed but not for poly(rA) *

oli-go(dT)-directed enzymeactivity. Such a

possi-ble defect in the intracellular virions of

IFN-treated cells would indeed make the virus

accumulation inthese cells much easierto

dem-onstrateby exogenous than by endogenous

en-zyme reactions. A third explanation could be

that thehigher activity of virions of IFN-treated

cellsmayreflectanincreased numberofenzyme

molecules per virion rather than virus

accumula-tion. This increased amountof enzyme is readily

detectable by exogenous reactions, inwhich a

vast excessoftemplate-primer is used.

Howev-er,since onlyone enzymemoleculepervirion is

supposedly involved in endogenous reactions,

this increased amount of enzyme per virion cannot be detected in such reaction conditions.

Effect of IFN on radioactivelabeling of

intracel-lular virions. To clarify which of the

above-mentioned possibilities indeed accounts for the discrepancy shown in Fig. 1B and C,

virus-producing cellswerelabeled for 48hwitheither

[3H]leucine or [3H]uridine in the presence or

absence of IFN. Labeled intracellular virions

were pelleted from the cytoplasmic fraction of

the cells and banded in 15 to 60% sucrose

gradients. Aliquots of each fraction of the

gradi-ent wereassayed for trichloroacetic

acid-precip-itableradioactivity. Figure 2A showsa remark-able accumulation of [3H]leucine-labeled intracellular virions in IFN-treated cells. This

finding eliminates the possibility that IFN treat-ment results in anincreased number ofreverse

transcriptase molecules per virion rather than virus accumulation, because such an increase wouldnotbe detected by[3H]leucine labeling of

o 12

J

z- 8

U) cr

'r

-r')

IJ

xQ

E

cj Cl >)

ESI

e

1.20 E 1.15 E 1.10

>--1.05

2

8

0 4 8 12 6 20 24

FRACTON NUMBER

FIG. 2. [3H]leucineand[3H]uridine labelingof

in-tracellular virions of IFN-treatedand untreated cells. Cells were labeled with [3H]leucine (A) and

[3H]uri-dine(B)for 48 h in thepresence(0)orabsence(0)of

IFN.Intracellular virions werepurifiedin 15 to60%o

sucrose gradients, and aliquots from each fraction,

collected from the gradients, were assayed for tri-chloroaceticacid-precipitableradioactivity.

(A xogenous Reaction

-I

I

II.,... ... ..,..

http://jvi.asm.org/

[image:3.496.53.244.89.418.2] [image:3.496.259.452.370.606.2]

(4)

virions. On the other hand, thisaccumulation of

intracellular virions could not be demonstrated in this particular experiment by [3H]uridine la-beling. It is therefore strongly suggested that the inabilitytodemonstrate intracellular virus

accu-mulationin IFN-treated cells by the endogenous

reverse transcriptase reactions wasdueto

defi-ciency of viral RNA rather thanto adefect in the

complex formation between p30 and reverse

transcriptase, although the latterpossibilitywas notdirectly examined and therefore couldnotbe totally excluded.

Effect ofIFN on viralgenomicRNA contentof intracellular virions. Sincereversetranscriptase

activity, measured inendogenousreactions,

de-pendsonboth thegenomicviral RNAservingas atemplate andon a primertRNA, the reduced endogenousenzymatic activityof the virions of IFN-treated cells could resultfromdeficiencyof

anyof thesevirus-packaged RNAspecies. Also,

the results of [3H]uridine labeling could not

provide any indication to suggest which of the RNA species was missing in the assembled

virions. To clarify this problem, the unlabeled

virions banded inthe sucrosegradients

present-ed in Fig. 1 were pelleted, and viral RNA was

extracted by chloroform-isoamyl alcohol and concentrated by ethanol precipitation. This

RNA was centrifuged through a 15 to 30%

sucrosegradient. Fractions werecollected and,

afteradditionof 50,gofE. coli tRNApermlas acarrier, theRNAof each fractionwas

concen-tratedby ethanol precipitation and analyzed for viralgenomic RNA sequences by hybridization

to MLV-specific

[3H]cDNA.

A major 70S peak

ofhybridizable RNA wasfound together witha

broad spectrum of smaller hybridizable RNA molecules, which are probably a degradation product of the 70S RNA (Fig. 3). More

impor-tant is that, although the amount of virions collected from thegradientof IFN-treated cells

was morethan threefold higher than that of the untreatedcontrol,nosignificantquantitative dif-ference could be detected between the

hybridi-zable RNA of the virions of IFN-treated and

untreated cells. It is therefore evident that the RNAdeficiency of intracellular virions of

IFN-treated cellsinvolves thegenomic viralRNA.

Effect of IFN on 4S RNAcontent of

intracellu-lar virions. To determine whether the RNA

deficiency of intracellular virions of IFN-treated

cells was restricted only to the viral genome,

RNA wasextractedfromthe

[3H]uridine-labeled

virionsof the experiment presented in Fig. 2B,

and the same amounts ofRNA (in counts per

minute) from virionsofIFN-treated and untreat-edcells wered analyzed by gelelectrophoresis.

Figure 4 shows that the ratio of 4S RNA/35S

RNA ofthe controlvirions(Fig. 4A)was consid-erably higher than that of virions from

IFN-treated cells (Fig. 4B). Since RNA of both

virions was similarly treated, it is unlikely that

this difference can be attributed tovariation in

degradation of 35S RNA during its preparation.

Since similar results were obtained in several

repeated experiments, it seems that IFN treat-ment results in deficiency of 4S RNA that is

evenstrongerthan thatof the viralgenome.

Fate of intracellular virions in IFN-treated and

untreated cells.Tofollow the fate of intracellular

virions underconditions ofanIFN-induced anti-viralstate,cellswerelabeledfor24hwitheither

[3H]leucine or [3H]uridine in the presence or

absence ofIFN. At the endof thislabeling, the cells were washed four times and further

incu-bated with nonradioactive medium. During this

additionalincubation,IFNcontinued to be

pres-ent in the medium of IFN-treated cultures. At

varioustimes afterremovaloflabels, extracellu-lar andintracellular labeled virionswereisolated

bysucrosegradient bandingandquantitated by

trichloroaceticacidprecipitation. Total incorpo-ration of labelsinto macromoleculeswas

deter-mined by trichloroacetic acid precipitation of cellaliquots. No increase in radioactive labeling

of cellular macromolecules occurred after label removal (Fig. 5A). Moreover, the cytoplasmic pool sizes of both labels were determined by

4°- 70S

28

S

18

S

40

z

30-~0

20

ol

0 4 8 12 16 20

FRACTION NUMBER

FIG. 3. Viral genome sequenceanalysis of intracel-lular virions of IFN-treated (0) and untreated (0)

cells. Unlabeled intracellular virions, banded in the

gradients presented in Fig. 1,werepelleted, and their RNAwasextractedbychloroform-isoamylalcohol and concentratedby ethanolprecipitation. TheRNA was

centrifugedthrough15 to30%osucrosegradients. Each

fraction, collected from the gradients, received50,ug of E. coli tRNA per ml and was precipitated with ethanol; the precipitates were hybridized with 2,500 cpmofMoloneyMLV-specific

[3H]cDNA.

Theextent of hybridization was determined by the fraction of

input [3H]cDNA rendered resistant to Si nuclease.

This [3H]cDNA showed 2.7% self-annealing, which

wassubtractedfrom each value.

J. VIROL.

http://jvi.asm.org/

[image:4.496.263.434.387.545.2]

(5)

RNA-LACKING CELLS

°x

₂₀

0~4

I I I I

> (B)35S

04 28S 18S 4S

3-2

0

0 10 20 30 40 50 60 70 80

SLICE NUMBER

FIG. 4. Sizeanalysisof RNAencapsidatedin intra-cellular virions ofIFN-treated and untreated cells. [3H]uridine-labeled intracellular virions, bandedinthe gradients shown in Fig. 2B, were sedimented, and RNAwasextracted asinthe legend toFig. 3. Equal

amounts of labeled RNA (in terms of counts per minute) from control virions (A)and from virions of IFN-treated cells (B)weresubjectedtogel electropho-resisasdescribedinthetext.The gelsweresectioned into 2-mmslices, whichwereextractedby 30% H202 andcounted inInstagel scintillation liquid,with

cor-rection forquenching.

directfiltering of cell aliquots without

trichloro-acetic acid treatment and subtracting from the

radioactivity of these aliquots the values of

trichloroaceticacid-precipitable radioactivity of

parallel aliqots. It was found that these pools

were promptly depleted after the cells were

washed(datanotshown). It is thusapparentthat

this wash was efficient for stopping further

sig-nificant incorporation of both labels into

pro-teins or RNA. Therefore, even if some new

labele&

virions ofprelabeledprecursors

contin-ued to be formed after removal of labels, they

would constitute only a small fraction of the

overall number of radioactive intracellular

viri-ons and therefore wouldnotraise serious

com-plications in interpreting the results. Both

[3H]uridine- and [3H]leucine-labeled

intracellu-lar virionsdisappeared from the control cellsata

rapid but approximately equal rate, which was

accompanied by an almost parallel appearance

of extracellular labeled virions (Fig. 5B and C).

(The appearance of only [3H]leucine-labeled

extracellular virionsisshowninFig. SD.) On the

other hand, in IFN-treated cells,

[3H]uridine-labeled intracellular virions disappeared at a

very slow rate (Fig. 5B), probably as a result of

the IFN-induced inhibition of their release.

However, the [3H]leucine-labeled virions dropped in these cells suddenly to about 35% of their initial level after a limited period of steady

state, and then they continued to decline at a

rate approximately equal to that of

[3H]uridine-labeled virions (Fig. 5C). It should be

empha-sized thatthe sudden fall in [3H]leucine-labeled

(A)

z15

9 15

M

6

cr8

10

0~~~~~

~'(B)

15£

5Q 0

> 0~~~~~

3f~0(5

3

0

cc

cr7

a-0

(D)

20~~~

'~i

AA

0

0 2 4 6 8 10

X TME AFTERLABEL REMMAL(h)

FIG. 5. Fateof labeled intracellular virions of IFN-treated and untreated cells. Cells were labeled with

[3H]uridine and [3H]leucinefor 24 h in the presence

(A)orabsence(0)of IFN. At the end of labeling the cellswerewashedfour times and received unlabeled fresh medium, with IFN addedtothemedium of the IFN-treated cultures. Atvarious times of incubation with the unlabeled medium, incorporation of labels into total trichloroacetic acid-precipitable materials wasdetermined in cell aliquots (A). Intracellular la-beled virionswere harvested from the

postmitochon-drial fraction of thecells(B andC) and extracellular virions wereharvested from the culture medium(D) andpurified by 15 to60%osucrose gradients. Values arethe total trichloroaceticacid-precipitable radioac-tivity in the 1.12- to 1.16-g/cm3band of these

gradi-ents.

http://jvi.asm.org/

[image:5.496.56.251.69.310.2] [image:5.496.262.449.210.525.2]

(6)

intracellularvirions ofIFN-treated cells wasnot

accompanied by acorresponding appearance of

labeled extracellular virions (Fig. SD). These

findings strongly suggest that most of the

RNA-deficient intracellular virions accumulated with-in IFN-treated cells collapse before being

re-leased from the cells. Furthermore, the slow

decline of[3H]uridine-labeled intracellular

viri-ons of IFN-treated cells, which was almost

parallel to the rate of virus release from these

cells,indicates thatthe RNAdeficiencyofthese

virionswas not due to itsdegradationafter being

encapsidated, butmorelikelyto adefectinvirus

assembly.

DISCUSSION

IFNis knowntointerfere with the replication of retroviruses(9,21).However,thetargetofits

actionseemsto varyfrom onesystem to anoth-er.Forinstance, inexogenousinfections, it has been found to inhibit someearly events before theintegration oftheproviralDNAintothehost

genome (2-4, 7, 31). In chronically infected

cells, IFN has been found to interfere with

various late steps, such as posttranslational

processing of viral precursor proteins (14, 29,

33,39),virusassembly (8, 15,32,38),oritsfinal release from the cell surface (10-12, 15). Since unlike mostothersystemsofretrovirus-infected cellsMoloneyMLVtypeCparticleswerefound

to be formed intracellularly in our chronically infected NIH 3T3 cells, apparently by budding into cytoplasmicvacuoles(2a), it was ofinterest

toinvestigate in detailthe effectof IFN on our

system. Inaccordancewith ourpreviousreports

(2a, 34), IFNwasfoundtostrongly inhibit virus release, and this inhibitionwasaccompanied by aremarkable accumulation of intracellularvirus

particles. However, our present data

demon-stratethatmostoftheintracellular virions

accu-mulating in IFN-treated cellsareRNAdeficient. This deficiency does not seem to result from a

degradation of the encapsidated RNA during

storage ofthe intracellular virions within

IFN-treated cells, but more likely from a defect in

virusassembly. It is interesting to mention that

formation ofRNA-deficient MLV particles has

been observed also in actinomycin D-treated,

virus-producing cells (22, 24-27). However,

whereas the RNAdeficiency observed in these

cells could be attributedtotheinhibition of viral

RNAsynthesis,in our cells IFN has beenfound

to have no effect on viral RNA synthesis (1).

RNAdeficiencyin oursystem involvedgenomic

viral RNA. Moreover, unlike the actinomycin

D-induceddeficiency, which is restricted to the

70S viral RNA(25, 26), thedeficiency imposed

in our systemby IFN was even stronger for the

small RNA species usually packaged in

retro-virus particles. Nevertheless, this uneven

defi-ciencyof the various RNAspeciesiscompatible withthe observationof Levin and Seidman(26)

thatpackaging oftRNA in MLV isindependent

of that of viral genomic RNA. Assembly of

reverse transcriptase invirus particleshas been

found also to be independent of packaging of

viral RNA (22, 27). This could be the reasonfor

our ability to demonstrate the accumulation of

intracellular virionsin IFN-treated cellsby

mea-suring their reverse transcriptase activity in an

exogenousreaction despite their RNA

deficien-cy. However,thisRNAdeficiencycould lead to

someadditionaldefects in the morphogenesisof

the virions,as reported by Jamjoomet al. (24).

Thesedefects could accountfor the breakdown

of the RNA-deficient intracellular virions

ob-served in ourIFN-treated cells. It isimportant

to notethat our data suggest that

RNA-contain-ing intracellular virions are rather stable, and it

is quite possible that the residual virus release

from IFN-treated cells involvesonlythiskindof

particle. Support for this proposal can be

provid-ed by our earlier observation (5) that IFN

inhib-its the relase of reversetranscriptase-containing

virus particles andinfectious virions to more or

less the same extent.

ACKNOWLEDGMENTS

Thisstudy wassupported by a grantfromtheChernow and StreinFoundation oftheIsraelCancerResearch Fund, New York,N.Y.

LITERATURE CITED

1. Aboud, M., R. Kimchi, M.Bakhanashvlli,andS. Salzberg. 1981.Intracellular production of virus particlesandviral components in NIH/3T3 cells chronically infected with Moloney murine leukemia virus: effect ofinterferon.J. Virol. 40:830-838.

2. Aboud, M., D. Moldovan-Levin, and S. Salzberg. 1981. CytoplasmicviralDNAsynthesis inexogenousinfection ofmurine leukemia virus: effect of interferonand cyclo-heximide. J. Virol. 37:836-839.

2a.Aboud, M.,R.Shoor,S.Bari,Y.Hassan,Z.Malik,andS. Salzberg.1982. Biochemicalanalysisand electron micro-scopic study onintracellular virions in NIH/3T3 mouse cellschronicallyinfected withMoloneymurine leukemia virus:effectof interferon. J. Gen. Virol.62:219-225. 3. Aboud, M., R. Shoor, and S. Salzberg. 1978. Effect of

interferononexogenousmurine leukemia virusinfection. Virology 84:134-141.

4. Aboud,M.,R.Shoor,andS.Salzberg.1980. Aneffectof interferonontheuncoatingof murine leukemiaVirusnot relatedtotheantiviralstate.J.Gen. Virol.51:425-429. 5. Aboud, M., 0. Weiss, and S. Salzberg. 1976. A rapid

quantitation of interferon withchronically oncornavirus-producingcells. Infect. Immun. 13:1626-1632.

6. Aboud, M., M. Wolfson, Y. Hassan, and M. Huleihel. 1982.Rapidpurification ofextracellular and intracellular Moloneymurineleukemiavirus. Arch.Virol.71:185-1%. 7. Avery, R.J., J.D.Norton, J.S.Jones,D.C.Burk,and A.G. Morris. 1980. Interferon inhibits transformationby murine sarcoma virus before integration of provirus. Nature(London)288:93-95.

8. Bandyopadhyay, A. K., E. H. Chang, C. C. Levi, and R. M. Friedman.1979.Structural abnormalitiesin murine leukemia viruses produced by interferon treated cells. Biochem. Biophys.Res. Commun. 87:983-988. 9. Billiau, A. 1977. Effect of interferon on RNA tumor

viruses. Tex.Rep.Biol. Med. 35:406-419.

J. VIROL.

http://jvi.asm.org/

(7)

10. Billiau, A., V. G.Edy, H.Sobis,and P. DeSomer.1974.

Influence of interferon on virus particles synthesis in

oncornaviruscarrier lines. II. Evidence foradirect effect onparticles release. Int. J.Cancer 14:335-340. 11.Bilhlau, A., H. Heremans, P. T. Aflen, J. De

Maeyer-Guingard, and P. DeSomer. 1976. Trappingof oncorna-virus particles at the surface ofinterferon treated cells. Virology73:537-542.

12. Bilhlau,A., H. Heremans, P. T. Allen, and P. DeSomer. 1976. Influence of interferon on the synthesis of virus particles inoncornavirus carrier lines. IV. Relevance to potentialapplicationofinterferon in infectious diseases. J.Infect.Dis. 133:A51-A55.

13. Bishop,M.J. 1978. Retroviruses. Annu. Rev. Biochem. 47:35-88.

14.Chang, E. H., and R. M.Friedman. 1977. Alarge glyco-protein of Moloneymurine leukemia virusderivedfrom interferon treated cells. Biochem. Biophys. Res.

Com-mun.77:392-397.

15. Chang, E. H., M. W.Mayers, P. K. Y. Wong,andR.M. Friedman. 1977. Theinhibitoryeffect of interferonona temperature-sensitive mutant ofMoloney murine leuke-miavirus.Virology77:625-636.

16. Chang, E. H., S. H.Mis,T.J.Triche,andR. M. Fried-man. 1977.Interferon inhibits mouseleukemia virus re-lease: an electron microscopic study. J. Gen. Virol. 34:363-367.

17. Dalton, A. J. 1966. Anelectron microscopic study of a virus induced murine sarcoma (Moloney).Natl. Cancer Inst. Monogr.22:143-168.

18. Dalton, A. J., F. Haguenau, and J. B. Moloney. 1964. Further electronmicroscopicstudies on themorphology of the Moloney agent. J. Natl. Cancer Inst. 33:255-275. 19.De Harven, E., and C. Friend. 1966. Origin of viremia in

murine leukemia.Natl. Cancer Inst. Monogr. 22:79-105. 20. Fan, H. 1978. Expression of RNA tumor viruses at the

translationandtranscription levels.Curr.Top.Microbiol. Immunol.79:1-41.

21. Friedman,R. M. 1977. Antiviral activity of interferon.

Bacteriol.Rev.41:543-567.

22. Gerwin, B. I., and J. G. Levin. 1977. Interactions of murineleukemiavirus corecomponents: characterization ofreversetranscriptase packaged in the absence of 70S

genomicRNA. J. Virol.24:478-488.

23. Ikawa,Y., M.Furusawa, and H. Sugano. 1973.

Erythro-cytic membrane specific antigensin Friend virus induced

leukemiacells.Bibl. Haematol (Basel)39:955-967.

24.Jamjoom, G. A., R. B.Naso, and R. B.Arlinghaus.1976.

Selective decrease inthe rateofcleavageof an intracellu-lar precursor to Rauscher leukemia virusp30bytreatment ofinfectedcells withactinomycinD.J. Virol. 19:1054-1072.

25. Levin, J. G., P. M. Grimley, R. M.Ramseur, and I. K. Berezesky. 1974.Defficiencyof60 to 70S RNA in murine

leukemia virus particles assembled in cells treatedwith

actinomycinD.J. Virol. 14:152-161.

26. Levin, J.G.,andJ. G. Seidman. 1979. Selective packag-ingofhost tRNAbymurineleukemia virusparticlesdoes notrequire genomicRNA. J. Virol.29:328-335. 27. Levin,J. G.,andJ.G.Seldnan. 1981. Effect of

polymer-ase mutations on packaging ofprimer tRNAP° during

murineleukemia virusassembly.J.Virol.38:403-408. 28. Luftig,R.B., J.F.Conscience,A.Skoultchi,P.McMillan,

M.Revel,andF.H. Ruddle.1977. Effect ofinterferonon dimethyl sulfoxide-stimulated Friend erythroleukemic

cells: ultrastructural and biochemical study. J. Virol.

23:799-810.

29. Maheshwari, R.K., D. K.Banerjee, C.J. Waechter, K.

Olden, and R.M. Friedman. 1980. Interferon inhibits

glycosylationof viralprotein.Nature(London)

287:454-458.

30. Monteralo, R.C., and D. P. Bolognesl. 1978. Structure andmorphogenesisoftype-Cretroviruses. Adv. Cancer Res. 28:63-89.

31. Moris, A. G., and D. C. Burk. 1979. An interferon sensi-tiveearlystep in theestablishment ofinfection of murine

sarcoma/leukemia virus. J. Gen. Virol.43:173-181. 32. Pltha, P. M., B. Fernie, F. Maldarelli, T. Hattman, and

N.A. Wivel. 1979. The error inassembly of MuLV in interferon-treated cells. J. Supramol. Struct. 12:35-46. 33. Pltha, P. M., B.Fernie, F.Maldarell, T. Hattman, and

N.A.Wivel.1980. Effect of interferon on mouse leukemia virus (MuLV). V. Abnormal proteins in virions of Rauscher MuLV produced in the presence of interferon. J.Gen. Virol. 46:97-110.

34.Salzberg, S., M. ahshvili, S. Bari, I.Berman,and M. Aboud. 1980. Characterization ofintracellular viral RNA ininterferon-treated cells chronically infected with murine leukemia virus. J. Virol.35:694-703.

35. Sato,T., C.Friend,andE. DeHarven.1971. Ultrastructur-alchanges in Friend erythroleukemia cells treated with dimethylsulfoxide. Cancer Res.31:1402-1417.

36. Sato, T., C.Friend, C.Stackpole, and E. De Harven. 1972. Coating of Friend leukemia virus after treatment with specific antiserum. Cancer Res.32:2670-2678.

37. Sugano, H., M.Furusawa,T.Kawaguchi,and Y.Ikawa. 1973. Enhancement oferythrocyticmaturation of Friend virus induced leukemia cells in vitro. Bibl. Hematol. (Basel) 39:943-954.

38. Van-Griensvan, L. J., M. E. Baudelaire, J. Peries, R.

Emanoll-Ravicovitch,and M. Bolron. 1971. Studies on the biosynthesis of murine leukemia virus RNA. Some prop-erties Rauscher leukemia virus from interferon treated JLSV cells, p. 145-153. In Le Petit coloquia. North-Holland, Amsterdam.

39. Yagi, M. J., N. W.King,Jr., and G. Bekesd. 1980. Alter-ations of mousemammary tumor virusglycoproteinwith interferon treatment. J. Virol. 34:225-233.

VOL.45,1983

http://jvi.asm.org/